Today’s New Reason To Believe

Fazale ‘Fuz’ Rana, Ph.D.

Rational Design of Novel Enzyme Highlights Biochemical Design

In 1978, three scientists (Hamilton Smith, Werner Arber, and Daniel Nathans) were awarded the Nobel Prize in Physiology or Medicine for the discovery of restriction enzymes and their applications. These proteins make genetic engineering possible. They have also contributed to the wave of advances that led to the sequencing of the human genome and to the emergence of other biotechnologies.

Restriction enzymes (or endonucleases) are important for another reason. They represent an interesting example of a chicken-and-egg biochemical system and comprise part of the collection of evidence that indicates life must stem from a Creator. Recent work on these proteins highlights this point.

Endonucleases are a family of proteins. This class of biomolecules cleaves DNA. Restriction endonucleases cut both strands of DNA at specific nucleotide sequences, called restriction sites. Specifically, restriction endonucleases protect the cell from foreign DNA, like viruses, by cutting the invading DNA into fragments.

These vital biomolecules occur in conjunction with proteins (called DNA methylases) that attach methyl groups to the same DNA sequences that would normally be cleaved by restriction endonucleases. When these sequences are methylated, restriction endonucleases cannot cut them. Restriction sites of the bacterial DNA are methylated to completely protect the bacterial DNA from being chopped up by its own restriction endonulceases. Foreign DNA, however, is not afforded this same protection.

DNA methylases and restriction endonucleases form a chicken-and-egg pair. Restriction endonucleases would destroy bacterial DNA without DNA methylases. On the other hand, if bacteria did not utilize restriction endonucleases there would be no need for DNA methylases. These two proteins are interdependent and must come into existence simultaneously.

New research by scientists from the Indian Institute of Science (Bangalore, India) helps demonstrate why biochemical systems like restriction endonucleases require the work of an intelligent Agent. These researchers performed experiments to understand the origin of restriction endonucleases from an evolutionary perspective. They also wanted to develop a strategy for engineering novel, nonnatural restriction endonucleases.

Evolutionary biologists think restriction endonucleases evolved from non-specific endonucleases through point mutations in the gene region that codes the DNA binding site on the protein surface. According to this model, once specificity was established recombination and genetic shuffling of the DNA sequences that encode the DNA recognition sites would have generated new restriction endonucleases with different specificities.

To explore this possibility the research team attempted to engineer a highly specific restriction endonuclease from one (R. KpnI) that promiscuously binds to DNA. To accomplish this goal, the scientists employed a rational design strategy to determine which amino acids in the R. KpnI structure to change. These workers had to make use of the detailed understanding of this protein’s structure and functional properties in order to develop the redesign strategy.

They successfully achieved their intended goal by replacing an aspartic acid residue with an isoleucine moiety at amino acid position 163 in the R. KpnI protein chain.

This research illustrates how carefully-thought-through single amino acid substitutions can alter the specificity of restriction endonucleases. This is important work that paves the way to engineer novel, nonnatural restriction enzymes that can expand the arsenal of tools available to molecular biologists and biochemists.

The researchers involved in this study also interpreted their success as support for the evolutionary origin of restriction enzymes with point mutations ushering in the first stage in the molecular evolution of these proteins. At first glance, this interpretation seems warranted.

Still, it’s important to keep in mind that the production of the highly specific restriction endonuclease from the original promiscuous protein required intelligent input from a team of highly trained biochemists who relied on the past work of other highly accomplished scientists. In a sense, this study empirically demonstrates that protein “evolution” requires the work of an intelligent Agent.

It’s also important to note that the researchers didn’t design the companion methylase protein. This protein isn’t necessary for most biotechnology applications. But without the methylase cohort, the reengineered restriction endonuclease would wreck havoc in vivo, destroying DNA that comprises the bacterial genome.

It’s very unlikely that a restriction endonulcease and its partner methylase would simultaneously appear in an evolutionary scenario. These coordinated events would require that changes in the restriction endonuclease would take place at exactly the same time as corresponding changes in the methylase. The only way for coordinated changes like this to happen is under the auspices of an intelligent Agent.

As I point out in my new book The Cell’s Design, human engineers frequently encounter chicken-and-egg problems when designing systems and processes. Everyday experience teaches that chicken-and-egg systems can come to fruition only through intentional planning and implementation. Chicken-and-egg systems, therefore, serve as a potent indicator of intelligent design.

I describe several other examples of chicken-and-egg systems in The Cell’s Design.

Posted by Fazale ‘Fuz’ Rana, Ph.D.

New Study Uncovers More Evidence for Biochemical Optimization and Intelligent Design

My new favorite drink is Coke Zero. I love how this zero-calorie soda tastes like the real thing, without any of the calories.

The close similarity between these two colas has spawned one of the more inventive advertising campaigns with the Coke big-wigs looking for a lawyer to sue themselves for taste infringement, since Coke Zero tastes so much like Coca Cola.

Sometimes two things seem to be indistinguishable, even though they really are quite different.

As I discussed last week, biochemists have recently come to recognize that biochemical synonyms—synonymous codons that specify the same amino acid in the genetic code—are actually distinct even though they have long been regarded as indistinguishable. It turns out that some codons are better suited than others for producing functional proteins. Biochemists refer to these preferred biochemical synonyms as optimal codons.

A new study demonstrates that the usage of optimal codons appears to be optimized as well, providing added evidence that life stems from the work of a Creator. Last week I presented the background information necessary to appreciate this new insight. This week I describe the research and discuss its implications.

Genetic Code

As I discussed last week, the genetic code constitutes a set of rules that translate the information stored in the nucleotide sequences of DNA into the amino acid sequences of proteins. Nucleotide triplets called codons represent the fundamental units of the genetic code.

Sixty-four codons make up the genetic code. Because the genetic code only needs to encode twenty amino acids, some of the codons are redundant. That is, different codons signify the same amino acid. In fact, up to six different codons specify some amino acids. Other amino acids are represented by only one codon.

Synonymous and Nonsynonymous Mutations

A mutation refers to any change that takes place in the DNA nucleotide sequence. Substitution mutations are one common type. When a substitution mutation occurs, one (or more) of the nucleotides in the DNA strand is replaced by another nucleotide. Sometimes substitution mutations generate a new codon that specifies the same amino acid as initially encoded. Biochemists refer to this type of change as a synonymous mutation. When a mutation produces a codon that specifies a different amino acid, it’s called a nonsynonymous mutation.

Nonsynonymous mutations can be deleterious if they affect a critical amino acid or if they significantly alter the chemical and physical profile along the protein chain. If the substituted amino acid possesses dramatically different physicochemical properties from the native amino acid, the protein folds improperly. This improper folding impacts the protein, yielding a biomolecule with reduced or even lost function.

Biochemists used to think that synonymous mutations had no impact whatsoever on protein structure, and hence function, since the amino acid sequence specified by the synonymous change would be identical.

Recently, biochemists have recognized that their views about synonymous changes were wrong. Even though the amino acid sequence doesn’t change, the protein structure can be altered. This altered structure stems from differences in the folding of the protein chain due to differences in the rate of protein production. Synonymous codons are read at differing rates. And the folding pattern will change depending upon the speed of protein construction.

Additionally, some synonymous codons are more likely to be misread by the cell’s machinery than others. So even though the information needed to make a particular protein isn’t changed, the wrong amino acid still may be introduced into the protein chain because of the codon used. This error, of course, can lead to structural and functional abnormalities in that protein, just as if a nonsynonymous change had occurred.

In either case, misfolded proteins can result. And this misfolding has disastrous consequences for the cell.

Misfolded Proteins

Misfolded proteins can cause profound problems for the cell. Their negative consequences extend beyond loss of function for the misfolded protein. Improperly folded proteins have a global impact on cellular health. These deformed proteins tend to form aggregates inside the cell, fouling up its inner workings. Biochemists think that many neurodegenerative diseases may have an etiology that involves aggregates formed from misfolded proteins. Misfolded proteins can also spread the misery to properly folded proteins. Once a protein has adopted a nonnative structure it can induce structurally-intact proteins to become improperly folded, enticing them to join the aggregated mess inside the cell. (As the biblical saying goes, “A little leaven, leavens the whole lump of dough.”)

In fact, even one improperly folded protein is enough to destabilize the ensemble of proteins inside a cell. (Go here for a technical article about the effect of misfolded proteins on global protein stability.) If all these problems weren’t enough, misfolded proteins can even disrupt cell membranes.

Optimal Codons are Optimally Distributed in Gene Sequences

Motivated by the impact of synonymous codons on protein folding, a team of biochemists conducted a study designed to explore the usage of optimal codons within genes and their relationship to protein folding. This research looked for correlations between codon usage and a number of parameters in a massive database of DNA sequences from a wide range of representative organisms, including the bacterium, E. coli; the yeast, S. cerevisiae; the nematode, C. elegans; the fruit fly, D. melangaster; the mouse, M. musculus; and humans.

The researchers discovered that critical amino acids tend to be specified by optimal codons. This makes sense, because errors in these positions will be much more detrimental than other positions in the protein chain. Mistakes in these positions are also much more likely to alter protein folding.

They also determined that proteins produced at relatively high levels in the cell have a greater fraction of optimal codons than proteins that occur at low levels. Again, this reflects an elegant strategy. The more often a protein is produced the more opportunity exists for protein misfolding to happen.

The biochemists also identified tissue dependence for the distribution of optimal codons. Tissues comprised of cells that are long-lived with a low turnover rate (like neurons) have proteins encoded by genes with a high fraction of optimal codons compared to rapidly replaced cell types. This again displays a remarkable biological logic. These types of cells would be susceptible to the accumulation of protein aggregates built up over the course of an organism’s life time. The increase of protein aggregates would not be much of an issue for short-lived cells with a high turnover rate.

One final note: The workers found these correlations in the data for all six organisms, suggesting that they have uncovered a universal pattern among all life-forms.

The bottom line: It looks as if the usage of optimal codons is optimized and appears to be undergirded by an impeccable biochemical rationale.

The Implications

Systems and objects produced by human designers are optimized. Optimization in an engineered system requires extensive planning and forethought and, therefore, stands as a hallmark of intelligent design. In fact, optimization is often synonymous with superior design.

As I describe in The Cell’s Design, life scientists have discovered that, like human designs, many biochemical systems are optimized according to a purpose. The usage of optimal codons marks just one more example.

The optimality of biochemical systems far exceeds the accomplishments of the finest human engineers and designers, in a way befitting a supernatural Creator. Biochemical optimization indicates that life must have materialized from the Divine Artist’s hand.

Biochemical design: It’s the real thing.

Posted by Fazale ‘Fuz’ Rana, Ph.D.

Scientists Create Enzyme from Scratch

As the business adage goes, “Time is money.” Time is also a valuable resource for living organisms. By themselves, most chemical reactions needed to sustain life occur at too slow a rate under physiological conditions to make life possible. Therefore, out of necessity, life’s chemical reactions are accelerated by special types of biological catalysts called enzymes.

These biomolecules are proteins specifically structured to speed up biochemical activities and operations. Enzymes are capable of increasing the rate of biochemical reactions by over a billion-fold in some cases! If not for enzymes, life would be impossible.

As I mentioned last week, a large team of collaborators recently published papers in Science and in Nature reporting on two enzymes created from scratch and capable of catalyzing nonbiological chemical transformations.

This work has several important implications: It helps biochemists to develop a better understanding of the relationship between enzyme structure and function. It also establishes an approach to generate novel enzymes which can have a wide array of practical applications. And finally, it affects attempts by life scientists to create artificial life in the lab, and, consequently, impacts the creation/intelligent design/evolution controversy.

Last week I provided the background information to appreciate this work. This week I want to describe the research and discuss its implications.

Though conceptually easy, designing these two enzymes was no trivial undertaking. The strategy employed by the researchers involved:

Modeling the reaction mechanism and the transition state of the reaction

Determining how to stabilize the transition state by placing chemical groups around the transition state complex

Designing an enzyme active site that yields the proper placement of chemical groups in space

Constructing the scaffolding of the protein chain to form and accommodate the active site

Fine-tuning the resulting enzymes

Executing this strategy required a large team of quantum and computational chemists, protein engineers, biochemists, and molecular biologists to create these biomolecules. The computations needed to design the active site and the initial enzyme architectures required hours and hours of supercomputer time.

It took so much effort to design the active site and protein scaffold primarily because the computational chemists and protein engineers weren’t able to build the enzymes from first principles. Instead they had to piece together the enzymes from the domains of about 100 proteins of known structure. They essentially mixed and matched protein regions, producing mosaic enzymes. Using this approach, they still had to sort through combinations for about 100,000 different protein regions. Once they created a scaffold that appeared to work, they had to optimize it using computational techniques. For one of the enzymes, this process yielded about 58 candidates.

Candidate enzymes were synthesized and evaluated in the lab as catalysts. Of the 58 possibilities only eight performed well enough to take to the next stage.

The structures of the best enzymes were then fine-tuned with in vitro evolution protocols. For one of the created enzymes, the in vitro evolution step improved efficiency by about two hundredfold.

Still, this enzyme operated with an efficiency that was ten thousand to a billion times less effective than enzymes typically found in living systems. According to the authors:

Although our results demonstrate that novel enzyme activities can be designed from scratch and indicate the catalytic strategies that are most accessible to nascent enzymes, there is still a significant gap between the activities of our designed catalysts and those of naturally occurring enzymes.

Even though the created enzymes fall short of those in nature, this advance truly represents a landmark accomplishment that stands as a towering intellectual achievement in every way. The ability to design enzymes that can catalyze novel, nonbiological chemical reactions will lead to better understanding of protein structure and enzyme catalysis. This methodology will also pave the way for protein engineers to design enzymes with industrial, agricultural, and biomedical utility.

This work also bears on the creation/evolution controversy. At first blush it appears as if scientists are one step closer to creating life in the lab. And if scientists can create life, where does that leave God?

In the face of this concern it’s remarkable to note how much effort it took to design a single enzyme that at best compares poorly with those found in nature. It took a collaborative effort from a large number of some of the finest minds in the world to develop and employ an effective design strategy. These researchers relied on sophisticated mathematical algorithms and technology (supercomputers and laboratory instruments) to carry out their scheme.

If it takes this much work and intellectual input to create a single enzyme from scratch, is it really reasonable to think that undirected evolutionary processes could routinely accomplish this task? And to a superior extent each time an enzyme emerges in nature?

It’s important to keep in mind that the simplest organism requires a few thousand different proteins to exist independently in its environment. How much effort would it take to construct the full range of enzymes needed for life, let alone design them to interact properly with each other? (For more details on life’s minimal complexity see Origins of Life and The Cell’s Design

In addition to the questions it raises about the origin of life, this new research provides direct experimental evidence that life’s molecules (and hence, life) must originate from the work of an intelligent agent, in this case a team of quantum and computational chemists, protein engineers, biochemists, and molecular biologists.

This recognition adds to the powerful case that can be made for intelligent design based on the features of biochemical systems. (See The Cell’s Design)

In light of this research, evolution seems to offer a poor return on investment. I’m investing my time and money behind the case for intelligent design.